p53 is a transcription factor that is activated by a variety of cell stresses and binds to a specific DNA sequence of a target gene, to control its transcriptional activity. p53 exhibits various physiological functions such as DNA repair, cell cycle arrest and apoptosis induction through the regulation of expression of a plurality of target genes.
p53 is usually maintained at a low level in a cell, and when the cell receives cellular stresses such as irradiation with radiations, irradiation with ultra violet ray, DNA damage by anticancer agents such as adriamycin, heat shock, osmotic shock and low-oxygen shock, p53 is stabilized and accumulated in the nucleus and activated as a transcription factor. Activated p53 induces the expression of genes such as p21WAF1 involved in checkpoint control, DDB2 and p53R2 involved in DNA repair, and BAX and p53AIP1 involved in apoptosis.
Recently, it has been revealed that modification after translations to p53, such as phosphorylation and acetylation, are profoundly involved in the regulation of expression of these physiological functions.
When minor DNA damage is caused by exposure to X-ray or UV ray, ATM (Ataxia Telangiectasia Mutated) and ATR (Ataxia Telangiectasia Related) are activated to phosphorylate Chk1 and Chk2 downstream therefrom, and Chk1 and Chk2 in turn phosphorylate serine at position 20 (herein after referred to sometimes as “Ser20”) in p53 and inhibit association of p53 with MDM2, thereby ubiquitinating p53 to prevent it from being decomposed with proteasomes, thus stabilizing p53. Serine at position 15 (herein after referred to sometimes as “Ser15”) in p53 is directly phosphorylated by ATM and ATR, thereby inhibiting the binding of MDM2 protein to p53, thus stabilizing and activating p53. The stabilized and activated p53 binds to a promoter of a G1 arrest period-related gene p21WAF1 or to a promoter of a DNA repair-related gene p53R2, to induce its expression.
When severe DNA damage is caused, Ser46 kinase is activated to phosphorylate serine at position 46 (herein after referred to sometimes as “Ser46”) in activated p53. The p53 phosphorylated at Ser46 binds to a promoter of p53AIP1 gene to induce expression of the p53AIP1 gene, thereby activating a p53-dependent apoptosis pathway to kill cells through apoptosis. Further, it is reported that p38MAPK activated by irradiation with ultra violet ray or with an anticancer drug phosphorylates serine at 33 position (herein after referred to sometimes as “Ser33”) in p53, thereby participating in p53-dependent apoptosis. It is also reported that both serine at position 376 (herein after referred to sometimes as “Ser376”) and serine at position 378 (herein after referred to sometimes as “Ser378”) located in the C-terminal region of p53 have been phosphorylated in a usual state in a cell, but when the cell undergoes DNA damage by irradiation with radiations, the phosphorylated state of Ser376 is maintained while Ser378 is dephosphorylated.
The expression of physiological functions of p53 is also controlled by acetylation of p53. p53 controls transcription of its target genes through interaction with transcriptional coactivators (cofactors) among which P300/CBP, PCAF, etc. have a histone acetyltransferase (HAT) activity. p53 interacts with such HATs to promote the histone acetylation of the target genes, while p53 itself is also acetylated by which lysine residues at positions 320, 373 and 382 (herein after referred to sometimes as “Lys320”, “Lys373”, and “Lys382” respectively) in the C terminal of p53. PCAF/GCN5 specifically acetylates Lys320, and p300/CBP specifically acetylates Lys373 and Lys382. When p53 is acetylated, its ability to specifically bind to DNA is enhanced. An estimated reason for this is that the positive charge of the C-terminal is neutralized by acetylation, which results in a structural change in p53 to cause exposure of a DNA domain of p53. There is also proposed a model in which the N-terminal is phosphorylated upon activation by DNA damage etc., followed by binding HAT such as p300/CBP to the N-terminal and acetylating the C-terminal.
Human p53 protein is composed of 393 amino acid residues and divided roughly into 3 regions, that is, (i) 1 to 100 amino acid residues in a transcriptional activation domain and a proline-enriched domain, (ii) 100 to 300 amino acid residues in a sequence-specific DNA-binding domain, and (iii) 300 to 393 amino acid residues in a tetramer-forming domain and a basicity-regulating domain, and it has been previously revealed that 7 phosphorylated sites (that is, Ser15, Ser20, Ser33, serine at position 37 (herein after referred to sometimes as “Ser37”), Ser46, threonine at position 81 (herein after referred to sometimes as “Thr81”), and serine at position 90 (herein after referred to sometimes as “Ser90”)) are present in the N-terminal region containing the transcriptional activation domain and the proline-enriched domain, and also that 5 phosphorylated sites (that is, serine at position 315 (herein after referred to sometimes as “Ser315”), serine at position 371 (herein after referred to sometimes as “Ser371”), Ser376, Ser378 and serine at position 392 (herein after referred to sometimes as “Ser392”)) and 3 acetylated sites (that is, Lys320, Lys373, Lys382) are present in the C-terminal region.
In the p53 gene whose mutations are detected most frequently in human cancers, most of the mutations are concentrated in DNA binding regions. This suggests that canceration by p53 mutation is attributable to an abnormality in its expression regulatory system. Accordingly, it is essential to elucidate the mechanism for regulation of expression of physiological functions by the above-mentioned modification after translation of p53, in order to elucidate the mechanism of canceration via p53 mutation, and there is strong demand for development of tools (for example antibodies) and measurement methods capable of detecting or quantifying phosphorylation and/or acetylation in specific sites of p53.
In view of such demand, methods of measuring the enzyme activities of Chk1, Chk2, ATM, ATR, and DNP-PK have been proposed, and p53 modification sites after translation specific polyclonal antibodies used in these methods are known (patent literature 1 to 5 and non-patent literature 1 to 18).
Patent Literature 1: International Publication No. 99/36532
Patent Literature 2: JP-A 2000-325086
Patent Literature 3: JP-A 2001-161398
Patent Literature 4: Japanese Patent Application Laid-Open (JP-A) No. 2002-524092
Patent Literature 5: JP-A 2003-93056
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